This invention relates to reagents useful as inhibitors of influenza virus replication and infection.
The following is a discussion of relevant art, none of which is admitted to be prior art to the pending claims.
Three types of influenza viruses (A, B, and C) are distinguishable by antigenic reactivities of their internal antigens. There are other biological properties which characterize the three types: (a) Influenza A viruses have been isolated from many animal species in addition to humans while influenza B and C viruses are mainly human pathogens; (b) the surface glycoproteins of influenza A exhibit much greater variability than their homologues in the B and C viruses; (c) morphological and molecular features of C viruses are distinctive from those of the A and B viruses.
The morphological characteristics of influenza viruses are a genetic trait, but spherical morphology dominates after passage in chicken embryos or tissue culture. The genes that specify morphology are uncertain, but segregate separately from the hemagglutinin (HA) and neuraminidase (NA) envelope surface proteins. Within the lipid envelope lies the matrix protein (M), which plays a structural function. Within the matrix shell are eight single-stranded RNA molecules of negative sense associated with the nucleoprotein (NP) and three large proteins (PB1, PB2, and PA) required for RNA replication and transcription. At least three viral encoded nonstructural proteins (NS1, NS2 and M2) are formed in infected cells.
The organization of the eight RNA segments within the virion has not been completely resolved. Although each segment may exist in vivo as a nucleoprotein complex, electron microscopic studies have shown that the internal component from disrupted virions is a single large helix. The virion particle does not seem to be a tight protective coat around the RNA because ribonuclease digestion of virion reduces the RNA segments to nucleotide. Genomic RNAs of influenza virus are held in a circular conformation in a virion and in infected cells by a terminal panhandle that plays a role in viral replication. The panhandle structure is present in all segments of genomic RNA.
The HA accounts for 25% of viral protein and is distributed evenly on the virion surface. It is responsible for attachment of the virus to cells and penetration of virus into cells early in infection. The HA monomer is encoded by the fourth largest RNA segment and is synthesized as a single polypeptide chain which undergoes posttranslational cleavage at a minimum of three sites. Cleavage of the HA polypeptide into HA1 and HA2 is necessary for virus particle infectivity. A sequence of 25-32 hydrophobic amino acids at the C-terminus of HA2 saves to anchor HA in the virus membrane. In spite of functional domain conservation in HA, the amino acid or nucleotide sequences of the proteins vary considerably between isolates of different subtypes.
The NA is the second subtype-specific glycoprotein of the virion and is composed of a single polypeptide chain. The NA is not evenly distributed on the surface of the virion but is found in patches. The role of the NA in the life cycle of the virus is unclear. No posttranslational cleavage of the NA polypeptide occurs. The nucleotide sequences of different NA gene isolates varies considerably between subtypes (e.g., A and B virus amino acid homology is 26-29%). The NA gene of influenza B encodes two proteins, NA and NB. The NA is thought to be structurally and functionally similar to the type A NA. The NB protein is a glycoprotein of unknown function which is 100 amino acids in length.
The nucleoprotein (NP) is one of the type-specific antigens of influenza viruses that distinguishes among the influenza type A, B, and C viruses. The NP is a multifunctional protein having a structural role in forming the nucleoprotein complex and a putative role in transcription and replication. Genetic analysis of a large number of influenza strains has revealed that the NP genes can be placed into one of five different groupings. All avian strains fall within two groups, equine strains fall within two more groups and all human and swine strains form the final group. The restriction of certain species strains to these groups suggests that the NP gene may influence species-specificity or host range.
RNA segment 7 encodes the two, M proteins (M1 and M2). The mRNA encoding M1 is colinear with RNA segment 7, whereas M2 is encoded by a spliced mRNA. The two proteins share the same initiation codon for protein synthesis and the eight amino acid residues before the 5' splice junction of the M2 mRNA. The remaining 88 amino acids of M2 are encoded in the +1 reading frame from nucleotides 740-1104. This organization of RNA segment 7 is present in all influenza A and B viruses sequenced.
The M1 protein is a virion structural protein that is intimately associated with the lipid bilayer in close proximity to both glycoproteins and the ribonucleoprotein complex. It is also believed to have a role in the down-regulation of the virion transcriptase activity. Passively transferred monoclonal antibodies to this protein do not confer resistance to infection by influenza virus.
The M2 protein of influenza A is an integral membrane protein that is expressed at the surface of infected cells. The M2 protein may be a virion associated protein with between 14 and 68 molecules per virion. Amantadine-resistant mutants of influenza virus contain mutations in the transmembrane domain of the M2 protein. Because amantadine alters viral penetration into cells, it is likely that M2 is in the virion.
Comparison of RNA segment 7 sequences of the H3N2 (Udorn) and H1N1 (PR8) strains show that the M protein coding sequences of these viruses (isolated 38 years apart) are highly conserved. Lamb, "The genes and proteins of influenza viruses," in. Krug. ed. The Influenza Viruses N.Y., Plenum. 1989. Comparison of 230 nucleotides of RNA segment 7 from 5 human H1N1, H2N2 and H3N2 strains isolated over a 43 year period suggests that the same segment 7 was retained throughout the antigenic shifts of HA and NA. Hall and Air, 38 J. Virol. 1, 1981.
Studies have shown that RNA segment 8 of influenza A and B encodes two nonstructural proteins which are translated from separate mRNAs. NS1 and NS2 polypeptides of influenza A share 9 amino acids at their N termini, after which the NS2 mRNA has a 423 nucleotide deletion; then, the NS2 mRNA rejoins the NS1 3' region in the +1 reading frame. NS1 is synthesized in large amounts early in infection. NS2 is made only late in infection. Both proteins share a nuclear localization signal and can be found in the nuclei of infected cells. Large deletions occur in the carboxyl termini of the NS1 proteins of field isolates from humans or birds, which indicates that a high degree of variation can be tolerated in this polypeptide without affecting its function.
The three largest proteins of the virion (PB1, PB2 and PA) are found associated with NP and virion RNA and carry the polymerase activity which transcribes invading viral RNA. The PB1 and PB2 proteins form a complex when expressed in the absence of other virion proteins or RNA and are probably required for complementary RNA synthesis. PA and NP are required for virion RNA synthesis. The PB1 gene of influenza B virus shows 61% homology with that of the influenza A virus.
Influenza virus produces an acute febrile infection of the respiratory tract characterized by abrupt onset prominent myalgias, headache and cough. Pneumonia is the most frequent complication; it may be primary viral (due to invasion of lung parenchyma), secondary bacterial, or mixed viral and bacterial pneumonia. It may be severe and progressive or mild and segmental. Other complications which occur with less frequency include Reye's syndrome, myocarditis, pericarditis, myositis, encephalopathy and transverse myelitis. It has been estimated that the direct costs of influenza exceed $1 billion per year and may reach $3 to $5 billion. Total costs may be two to three times higher.
Two types of vaccines are available for influenza. The "split" vaccines are chemically treated to reduce pyrogenic components and are the only type given to children under 13 years of age. The "whole" vaccine is generally given to adults. Protective antibody titers are present in more than 90% of normal subjects after vaccination with influenza A antigens, but there is much less response to influenza B antigens. Additionally, elderly subjects and patients with renal failures or immunosuppression are at much greater risk to infection even with vaccination. The 70-80% efficacy of the vaccine is only observed when strain matches are good. Lower efficacy is observed when the match is not close, and when patients are immunocompromised, or in institutional situations in which virus is readily transmitted.
Two drugs, amantadine and rimantadine, are as effective as influenza vaccine in preventing influenza A infections. Unfortunately, they are not as active against influenza B, which is responsible for 20% of all influenza epidemics and in a given year may be the only virus circulating. Amantadine is approved in the United States; rimantadine is not. Both drugs appear to impair the uncoating of viral RNA in infected cells by blocking the acidification process required to open the viral particles.
Resistance to amantadine and rimantadine is easily produced in the laboratory by serial passage of strains of influenza A virus in low concentrations of the drug, and such isolates are cross-resistant to both drugs. Drug resistant strains of influenza virus are able to initiate infection of cells as effectively as their wild type progenitors. Resistance is associated with the presence of point mutations in the RNA sequence coding for the M2 protein. This occurs most frequently at amino acid 31, but may also occur at positions 27 to 34, which encompass the transmembrane domain of the protein. It has been hypothesized that M2 protein may act as an ion channel to facilitate the acidification of the virus particle, and that amantadine and rimantadine block this viral-envelope pore.